KR20180100437A - New molecules with anticancer activity - Google Patents

Abstract

(I)The present invention relates to new compounds extracted from algae having the following formula (I) used for inhibiting the growth of cancer cells, and their extraction and separation methods.

(I)

Description

New molecules with anticancer activity

The present invention relates to a novel molecule extracted from marine algae, a production method thereof, and a use for inhibiting the growth of cancer cells.

Cancer is a disease that seriously compromises human health. Around the world, about 6 million people die of cancer each year, and another 10 million are seriously affected by the disease. According to the World Health Organization (WHO) estimates, in the 21st century, cancer will be the "highest death cause" of mankind.

Over the past several decades, various cancer treatments have become available, including surgery, radiation therapy, chemotherapy, hormone therapy, gene therapy and immunotherapy, and surgery, radiation therapy, and chemotherapy have become the primary means. Chemotherapy refers to treating cancer with chemicals. This is the fastest growing area of cancer treatment. Many new drugs targeting different targets have been prepared for clinical applications, and research and development of mechanisms of drug action and pharmacokinetic mechanics has created a suitable route of administration and means for killing tumor cells while protecting normal tissues.

Studies of natural derived molecules to inhibit cancer cells lead to the discovery of molecules such as taxol or vinblastine. Notwithstanding the focus at the treatment center and the availability of daily chalcogenide, there is a serious limitation to this treatment.

One major problem when treating cancer is obtaining selectivity for this type of cancer cell. There is a need to discover and isolate new powerful compounds with selective activity against certain types of cancer cells to provide highly selective anticancer molecules.

Against this backdrop, new molecules to add to existing chemotherapy drugs are highly desirable.

A key aspect to which the present invention addresses is to provide novel molecules extracted from Chaetomorpha Cannabina (CC) seaweeds.

According to a first aspect, concentrated extracts from the compound defined by formula (I) are provided.

(I).

(I).

According to a first aspect, there is provided a compound as defined by formula (I) in enriched or purified form.

According to another aspect of the present invention there is provided the use of an extract or a compound as defined herein for inhibiting the growth of cancer cells, for example, in vitro or in vivo in mammals.

According to another aspect of the present invention there is provided the use of an extract or a compound as defined herein for the manufacture of a composition for treating cancer in a mammal.

According to another aspect, the present invention provides a composition comprising an extract or a compound as defined herein, mixed with a physiologically acceptable additive.

According to another aspect, the present invention provides the use of an extract or a compound as defined herein for the manufacture of a composition for treating cancer in a mammal.

According to another aspect, the present invention provides a method of inhibiting the growth of cancer cells comprising connecting the cell with a growth inhibitory concentration of an extract, compound or composition as defined herein.

According to another aspect, there is provided a method of treating cancer in a mammal comprising administering to the animal an inhibitory concentration of growth of the composition, as defined herein.

According to another aspect, the present invention provides a method for separating a compound as defined herein, comprising the steps of:

a) Mixing the biomass of Chaetomorpha Cannabina (CC) seaweed with the first solvent to obtain biomass (solvent mixture)

b) eluting the mixture in a SPE column with a second solvent of varying concentration; Solvent fraction recovery

c) separating the methanol fraction from the b) step on a Combiflash column with various concentrations of a third solvent, recovering the fraction containing the compound of formula (I) and optionally concentrating or purifying the compound from the fraction

Figure 1. Photographs of Chaetomorpha Cannabina (CC) algae samples used for extraction.
Figure 2. In vitro activity of different concentrations of crude extract # 1 (NC77) on viability of 7 cell lines
Figure 3. Fractionation and subfractionation strategy of crude extract # 1 of Chaetomorpha Cannabina (Nc77)
Figure 4. In vitro activity of the crude extractant # 1 (NC77) fraction (F1-F5) on the viability of five cancer cell lines
Figure 5. GC chromatograms of FAME prepared from NC77 and NC77-Fr-4 fractions
Figure 6. ¹ H-NMR profile of NC77 extract
Figure 7. ¹ H-NMR profile of NC77-Fr-4 extract
Figure 8. HPLC comparison between two samples of NC77-Fr4 at two different collection days
Figure 9. Flowchart of fractionation, sub-fractionation and purification of the major composition from NC77
Figure 10. UPLC-DAD / ELSD / HRMS profile of compounds of formula (I)
Figure 11. HRMS spectrum of the compound of formula (I)
12. < 1 > H-NMR spectrum of the compound of formula (I)
Figure 13. COZY spectrum of compound of formula (I)
Figure 14. TOCSY spectrum of compound (I)
Figure 15. HSQC spectrum of the compound of formula (I)
Figure 16. HMBC spectrum of the compound of formula (I)
Figure 17. Decomposition ion observed in HRMS for the compound of formula (I)

The singular forms " a, " and " and " the " include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to " one cell " includes such a plurality of cells, and references to " the cultured tissue " include those equivalent to those known to one skilled in the art, do. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise specified.

As used herein, the term " about " or " approximately " To be accurate, for example, when used with: 90% means 90% ± 9%, that is, from 81% to 99%. More precisely, the term drug refers to + or - 5% of the indicated number, for example 90% means 90% +/- 4.5%, i.e. 86.5% to 94.5%. When used in the context of pH, the term " about " means +/- 0.5 pH units.

As used in this specification and the claims, the words " comprising " (and all forms of inclusions such as &Quot; (and all forms of inclusive inclusion such as " inclusive "), inclusive (and all forms of inclusive inclusion such as " inclusive ") are inclusive or unlimited, Do not exclude steps.

As used herein, the terms " disease " and " disorder " may be used interchangeably or that a particular disease or condition may be absent from a known causative agent (because the pathogen has not yet been identified) Although not yet recognized as a disease, it is only recognized as an undesirable condition or syndrome, and in that respect it is somewhat a specific set of clinicians-confirmed syndromes.

As used herein, the term " subject " or " patient " refers to an animal that is the subject of treatment, observation or experiment, mammals,

&Quot; Mammal " refers to non-domestic animals such as livestock such as humans and laboratory animals and pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits) .

The term " extract " as used herein refers to a composition prepared by connecting a seaweed biomass with a solvent according to the procedure of the present invention, and shows inhibitory activity against at least one cancer cell line in vitro. In one aspect of the invention, the extract shows inhibitory activity against cancer cell growth in vitro. As used herein, the term " extract " means an extract that is crude, fractionated, sub-fractionated, isolated, isolated, concentrated or purified, but is not limited thereto.

The term " isolated " is used herein to denote that a compound exists in a physical environment, distinct from that occurring in nature. For example, isolated molecules can be isolated much from the naturally occurring complex cellular environment as in crude extracts. When the isolated molecule is concentrated or purified (e. G., Concentrated or purified), the absolute level of purity is not critical, and those skilled in the art will be able to determine the appropriate level of purity according to the intended use of the biomass have. In some circumstances, isolated molecules form part of a buffer system or a composition that may contain other components (e. G., Some crude extract containing many other substances). In other circumstances, isolated molecules can be purified by NMR or chromatography (e.g., LC-MS) to the requisite homology, as revealed by using, for example, spectrophotometric methods.

The term " untreated " means a compound or molecule that is not completely separated from the components of the original composition in which it is present.

Thus, the term " separation ", " purification " or " isolation " refers to a method by one or more contaminating (i.e., unrelated) components of a biological sample that are removed from one or more other desired components of the sample.

The molecule (s) as described herein may contain additives such as pharmaceutically acceptable additives, pharmaceutically acceptable carriers and pharmaceutically acceptable vehicles, or additives, such as neatraceutical or nutraceutically acceptable additives, Or a formulation comprising a nutraceutical or a nutritional acceptable additive, such as a nutritionally acceptable vehicle.

The term " pharmaceutically acceptable " as used herein, when administered to a human, is physiologically tolerable and refers generally to allergic or similar undesirable reactions such as gastrointestinal disorders, vertigo, and the like. It shows the whole molecule and composition not produced. As such, the term "pharmaceutically acceptable" means that the substance has been approved for use by an animal, particularly a human being, in a federal or state government or a regulatory agency listed in the United States Pharmacopoeia or other generally accepted pharmacopoeia.

The term " carrier " means a diluent, adjuvant, excipient or vehicle that can be administered with the compound of the present invention. Sterile water or aqueous salt solutions and aqueous dextrose and glycerol solutions may be used as carriers, especially for injection solutions. Suitable pharmaceutical carriers are described in " Remington ' s Pharmaceutical Sciences " by E. W. Martin.

The molecule (s) and composition (s) of the present invention may be prepared as nutritional preparations such as foodstuffs including medical foods, functional foods and dietary supplements. &Quot; Medical or functional food " is defined as being ingested as part of a normal diet, but has physiological advantages and / or reduced basal nutritional function and the risk and condition of diseases such as chronic diseases. A " dietary supplement " is defined as supplementing a human diet, but is generally provided in the form of a pill, capsule, tablet, or the like. By way of example, and not limitation, dietary supplements may include one or more of the following ingredients. Dietary materials, concentrates, metabolites, components, extracts, or combinations thereof to supplement diets by increasing total dietary intake. Dietary supplements may be incorporated into foods, such as functional foods, designed to promote health or prevent disease or disability. When administered as a pharmaceutical preparation, the composition may be administered to the patient in any of a number of ways as a prophylactic or therapeutic method. The subject composition may be administered alone or in combination with other pharmaceutical agents, and may be combined with its physiologically acceptable carrier. The effective amount and method and purpose of a particular formulation may vary depending upon the individual subject, the stage of the disease or condition, and other factors evident to a person skilled in the art. In addition to the pharmaceutical prescription drug, the Nutraceutical preparation can also be monitored (e.g., plasma level monitoring) to maintain the desired level of concentration of the subject composition during the course of treatment.

The term " nutraceutical " is used to denote any substance that is part of a food or food product and provides medical or health benefits, including prevention and treatment of disease or condition. Thus, the Nutraceuticals are products isolated or refined from foods that are generally sold in a medicinal form that is not associated with food. Nutraceuticals have physiological benefits or provide protection against chronic diseases. For this reason, compositions under the trademark "Nutraceutical" range from isolated nutrients, dietary supplements and certain diets to foods produced by genetic engineering, herbal products and processed foods such as cereals, soups and beverages . In a more technical sense, this term has been used to refer to products separated or refined from food and is usually sold in a form that is not generally associated with food, generally in a medicinal form, . Suitable nutraceutically acceptable additives may include solutions such as solutions containing vegetable and / or animal and / or fish oil.

The terms " molecule " and " compound " are used interchangeably herein.

Targeted as an alternative source of anticancer molecules, anticancer compounds extracted and isolated from solvent extracts of Chaetomorpha Cannabina (CC) are provided.

extract

According to a particular embodiment of the present invention, bioactive molecules in the seaweeds listed above have been found in 100% MeOH moieties and are defined by the following formula:

(I),

(I),

Active molecules from the extract

According to a particular embodiment, the present invention provides a compound defined by the formula (I)

(I),

(I),

 As its racemic mixture or as an enantiomer.

Composition

According to a particular embodiment of the present invention, there is provided a composition comprising a compound as defined herein, in admixture with a physiologically acceptable additive.

How to use and use

In accordance with alternative embodiments, the present invention provides the use of a compound as defined herein to inhibit the growth of cancer cells. In particular, for the preparation of a composition for treating cancer in a mammal, the use of an extract as defined herein is provided.

According to an alternative embodiment, the present invention provides the use of a compound as defined herein for treating cancer in a mammal.

According to a particular exemplary embodiment, the present invention provides a method of inhibiting cancer cell growth, comprising contacting said cell with a growth inhibitory concentration of a compound or composition as defined herein.

Treatment method

In particular, there is provided a method of treating cancer in a mammal comprising administering to said mammal a growth inhibitory concentration of a composition as defined herein. In particular, the mammal is a pet animal or a human.

Extraction and Separation Methods

According to a particular embodiment, there is provided a method of extracting a compound as defined herein, comprising the steps of:

a) Mixing the biomass of Chaetomorpha Cannabina (CC) seaweed with the first solvent to obtain biomass (first solvent mixture)

b) eluting the mixture in a SPE column with various concentrations of a second solvent, and recovering the second solvent fraction

c) sorting the above methanol fraction from step b) on a Combiflash column at various concentrations of the third solvent mixture, recovering the fraction containing the principal component

Depending on the alternative formulation, in addition to the process of the invention, a hexane-degreasing step precedes step a).

Depending on the particular model, the method further comprises the following steps. d) Preparing the fraction on the semi-preparative HPLC column with a fourth solvent mixture to obtain a concentrated lower fraction of Compound (I)

Depending on the selection scheme, the method further comprises the following steps. d ') < / RTI > by concentrating or purifying the compound of formula (I)

(I).

(I).

In accordance with the alternative convention, the method further comprises the following steps. e) drying the above sub-fractions by removing the solvent to obtain a concentrated dry extract from the compound of formula (I)

(I).

(I).

Solvent for extraction

In particular, the molecules are extracted with a first solvent. More particularly, the first solvent of the extract is water or an alcohol. More particularly, it is aqueous ethanol.

In particular, the crude extract is an aqueous ethanolic extract of CC. In particular, the crude extracts are aqueous ethanol extracts of 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%. Even more particularly, the crude extract is 80% aqueous ethanol extract. Most particularly, the crude extract is a previous hexane-defatted extract.

In particular, the second solvent is methanol, aqueous methanol or a mixture of methanol and acetonitrile. Particularly, the extracts contain 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% MeOH or 100% MeOH or CH2Cl2: MeOH (1: 1). Most particularly, this fraction is from 30% aqueous MeOH to 100% MeOH fraction.

In particular, the third solvent is methanol or aqueous methanol. Most particularly, the extract is a flash column sub-fraction of the C-18 fraction. In particular, the lower fraction is about 40% MeOH sub-fraction.

The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and that the inventors contemplate to limit the scope of the invention, It does not attempt to indicate that it is all or only an experiment. Efforts have been made to ensure accuracy of the numbers used (eg, quantity, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weights are weight average molecular weights, temperatures are degrees Celsius, pressures are atmospheric or near atmospheric.

Example - Antitumor activity of compound (I) purified from marine algae harvested in Newfoundland and Labrador

Example 1 - Seaweed collection and identification

The collection program for seaweeds has been established over several hours for several geographic areas in Newfoundland, Labrador and Quebec.

The general collection procedure was as follows. While the scuba diver was collecting algae from the lake, the algae were collected manually by a knife in the intertidal zone. The sample was placed in a plastic sampling bag and transferred to the laboratory from the seawater cooler. Upon arrival in the laboratory, the various items were washed individually to remove coagulants and adherents (sand, mussels, etc.). Then, the sample was visually inspected to make sure it was clean. If not, the residue was hand washed with additional washing. The seaweeds were wiped dry, weighed to the nearest g (wet weight of the plant) and chopped. The finely divided biomass was transferred to an Erlenmeyer flask and frozen at -60 ° C until the extract was prepared.

A representative sample of each of the other samples of Chetomorpha cannabina was also photographed (see FIG. 1) and frozen at -20 ° C to confirm species by Dr. Robert Hooper, Bird Scientist at Newfoundland Memorial University.

Example 2 Extract preparation

2.1 Freeze drying

To prepare the sample for extraction, the seaweed was first lyophilized. An Erlenmeyer flask containing frozen chopped seaweed at -60 ° C was placed in a freeze dryer and lyophilized under reduced pressure for 72 to 96 hours at 69 x 10 ^-3 mbar. The weight (g) of the dried biomass was then reported as plant dry weight (g). This step describes the difference in moisture content in seaweeds that may affect the solubility of the bioactive component. Secondary plant metabolites are also more stable than when stored in dry form. Moreover, large-scale extraction of dried plant biomass can cause less problems than extracting fresh biomass. To preserve thermally labile compounds, low temperature conditions are used throughout the extraction process.

2.2 Defatting the sample

It is known that the lipid fraction of seaweed varies from 1 to 5% of the predominantly polyunsaturated fatty acid algae. While green algae possess levels of alpha-linolenic acid (n3, C18: 3), brown algae and flounder algae are abundant, especially long chain polyunsaturated fatty acids such as eicosapentaenoic acid (n3, C20: 5). Because these polyunsaturated fatty acids are extremely susceptible to oxidation, they can produce lipid oxidation products during analysis. Samples were degassed prior to compound extraction in order to remove the oxidation process above which could affect the results.

The lyophilized seaweed sample was ground into powder and degreased at room temperature by mixing the powder with hexane (1: 5, w / v, 5 minutes) in a waring mixer. The degreased sample was air dried, vacuum packed in a polyethylene pouch, and kept at-20 C until extraction.

2.3 crude extraction

Other solvents or solvent systems may be used for the extraction of the compounds. Ethanol is commonly used because it is less toxic than other solvents. Moreover, ethanol extracts showed the highest antioxidant activity in many studies.

The compounds were extracted with 80% aqueous ethanol for 24 hours at 46 < 0 > C. Subsequently, the solvent was removed under vacuum at 37 占 폚 for 45 minutes and the resulting concentrated slurry was freeze-dried at -80 占 폚 and 69 占 폚 -10 ^-3 mbar using a freeze dryer at 72 to 96 hours under reduced pressure. The dry extract was weighed (dry weight of extract from g) and stored at -60 ° C until production for inspection.

The extraction yield was calculated and expressed as g of dry extract per gram of dry seawater according to Table 1.

Extraction yield Bell seabird Date of collection area Extract dry weight (g) Yield (dry extract g / dry plant g) Chaetomorpha
cannibina
(Extract # 1 for NC 77)
green July 22, 2013
Rocky Hr.,
Netherlands 4.88

Chaetomorpha
cannibina
(Extract # 2 for NC 107)
green August 26, 2014
Mutton
Bay, Quebec 0.7 3.88%

Example 3 Anticancer test of crude extract of seaweed

3.1 Purification through biological activity tracking fractionation

Initially, seaweed extracts were evaluated for their anticancer activity in in vitro models via CIMA analysis. From these results, the extract with the greatest anticancer potential was selected for purification through bioactive trace-fractionation.

3.2 Compound Preparation

The mother liquor of the extract was prepared in 10 mg / mL dimethylxhosis (DMSO) and stored in 200 μl aliquots at -20 ° C. until analysis. This preparation ensures that the DMSO delivered to the cultured cells never exceeds 1%.

3.3 CIMA analysis

The extracts produced were evaluated according to long term exposure conditions in which the cells were seeded at 2 x 10 ³ cells / wall (96 wells-plate) and incubated with the test compound for 72 hours. Each compound was evaluated over the concentration range (0, 10, 25, 50 or 100 μg / ml). Cell proliferation was initially assessed using standard colorimetric indicators of metabolic activity (CIMA) analysis. In this assay, tetrazol reduction was assessed as a measure of metabolic function assessing mitochondrial activity to determine the extent of cell proliferation in cultured tissues. This assay, which causes altered color to give a change in absorbance, is based on the reduction of yellow tetrazolium salt in purple formazan by mitochondrial reductase in living cells. The following six human cell lines were selected for the first round of evaluation. (Fibroblast, non-cancerous) (Fig. 2 and Fig. 3), and the tumor cells of U373 (syngeneic astrocytoma), A549 (lung cancer species), THP-1 (acute mononuclear leukemia), MCF7 (breast adenoma), SKOV3 Table 2 shows the IC ₅₀ for each extract.

From the results shown in Table 2, Extract # 1 (NC77) was selected for further fractionation and evaluated in the same manner.

In vitro screening of fractions # 1 (NC77) and # 2 (NC107)

Cell line (ug / ml) Extract # 1 Extract # 2 One NC107 Average Standard Average Standard A549 100 67.5 1.6 70.5 2.5 50 77.1 2.4 88.8 3.2 25 78.8 3.1 91.3 3.6 10 81.7 2.7 93.2 3.6 One 94.6 4.6 102.5 6.8 0 100.0 0.0 100.0 0.0 DMSO 1% 92.5 3.1 92.5 3.1 SDS 5% 68.7 6.6 68.7 6.6 PC3 100 82.5 2.2 81.1 3.0 50 77.2 4.0 87.1 2.3 25 86.4 3.4 90.3 2.8 10 91.7 2.0 94.9 0.8 One 98.2 1.3 97.9 5.0 0 100.0 0.0 100.0 0.0 DMSO 1% 87.0 4.0 87.0 4.0 SDS 5% 77.3 13.3 77.3 13.3 MCF7 100 45.6 1.0 48.7 4.5 50 60.3 2.6 71.2 4.9 25 72.7 3.6 88.5 8.4 10 78.7 3.9 94.0 8.5 One 101.8 12.8 102.7 3.8 0 100.0 0.0 100.0 0.0 DMSO 1% 94.7 6.2 94.7 6.2 SDS 5% 44.1 8.0 44.1 8.0 SKOV3 100 50.9 4.6 45.1 5.6 50 72.3 3.7 67.8 7.4 25 79.8 4.8 76.4 5.9 10 84.7 4.0 79.5 6.2 One 95.5 5.7 93.6 9.5 0 100.0 0.0 100.0 0.0 DMSO 1% 95.3 7.5 95.3 7.5 SDS 5% 78.9 3.9 20.0 78.9



U373 100 48.4 2.0 34.5 3.1 50 55.7 3.0 54.4 2.4 25 57.0 2.8 61.3 4.4 10 61.8 4.6 76.2 4.6 One 94.6 2.7 96.6 6.9 0 100.0 0.0 100.0 0.0 DMSO 1% 83.4 7.7 83.4 7.7 SDS 5% 48.4 6.4 48.4 6.4



THP-1 100 40.8 1.2 35.8 1.4 50 52.2 1.7 47.7 1.8 25 67.9 4.4 56.6 4.8 10 88.2 3.7 82.4 9.4 One 99.4 7.2 105.6 5.8 0 100.0 0.0 100.0 0.0 DMSO 1% 89.2 3.6 89.2 3.6 SDS 5% 20.3 4.0 20.3 4.0



CCD1079
SK 100 60.3 4.6 46.1 1.1 50 65.5 4.5 55.7 1.4 25 67.3 3.6 62.7 2.7 10 82.8 7.6 76.5 4.0 One 100.3 10.3 92.8 3.1 0 100.0 0.0 100.0 0.0 DMSO 1% 91.3 7.8 91.3 7.8 SDS 5% 9.4 0.9 9.4 0.9

IC50 calculations for crude extract NC77 and NC 107 NC77 NC107 A549 - - PC3 - - MCF7 81.9 95.9 SKOV3 - 88.2 U373 66.l 50.8 THP1 75.9 45.1 CCD1079SK - 86.6

- LD ₅₀ > 1-μg / ml

3.4 fractionation

15 mL of each of 5% methanol (fraction 1), 25% methanol (fraction 2), 50% methanol (fraction 3), 100% methanol (fraction 4) and methanol: dichloromethane 1: The fractions were separated into 5 fractions by C-18 SPE column separation. The extract was fractionated and the resulting fractions were evaluated using CIMA analysis. Biologically active trace fractions provided detailed information on specific biologically active compounds.

The following quantities were derived from the scheme described in FIG. 3 and summarized in Table 4.

3.4.1 Fraction Results

The resulting fractions were evaluated using CIMA analysis. Biologically active trace fractions provided detailed information on specific biologically active compounds. (Table 5) Table 6 shows IC50 of each fraction.

In addition, as shown in Figure 4, activity is present in fractions 3 and 4. Given the yields obtained for each fraction, the fractionation results clearly showed that the anticancer activity was distributed in F4, and the selection priority for Extract # 1 (fraction 4) was established. Thus, fraction 4 (0.34 g) from NC77 was further subjected to sub-fractionation as shown below.

Identification of active fractions from extract # 1 One
Cell line (ug / ml) F1 F2 F3 F4 F5 Average Standard Average Standard Average Standard Average Standard Average Standard SKOV3 100 92.4 8.9 91.4 2.3 51.3 7.0 50.1 * 5.8 100.8 10.8 50 104.2 12.5 92.6 3.5 77.0 11.0 53.8 # 3.4 96.3 4.3 25 100.0 6.3 94.0 0.6 87.5 9.7 60.7 * 6.2 95.1 12.1 10 101.0 11.1 99.5 3.9 91.4 10.9 68.3 * 4.7 93.9 12.9 One 103.6 8.2 102.3 5.3 100.9 12.3 95.0 2.5 108.4 9.9 0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 DMSO 1% 88.6 3.7 88.6 3.7 88.6 3.7 88.6 3.7 88.6 3.7 SDS 5% 56.5 5.4 56.5 5.4 56.5 5.4 56.5 5.4 56.5 5.4 MCF7 100 92.6 11.2 98.8 4.4 27.8 2.5 43.2 # 5.4 82.3 2.5 50 98.4 4.9 99.4 7.3 52.5 6.2 50.3 * 3.0 91.1 2.9 25 105.8 6.4 100.0 2.8 92.9 8.2 73.3 * 7.9 96.7 7.5 10 103.4 2.2 106.8 5.4 101.1 4.2 80.4 15.2 96.0 3.7 One 103.3 6.4 101.1 3.6 105.7 4.7 97.1 11.3 98.9 2.9 0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 DMSO 1% 92.1 5.9 92.1 5.9 92.1 5.9 92.1 5.9 92.1 5.9 SDS 5% 49.9 5.6 49.9 5.6 49.9 5.6 49.9 5.6 49.9 5.6 U373 100 81.1 3.6 69.0 6.3 49.2 3.1 60.7 4.2 72.2 1.9 50 85.3 8.4 82.3 5.9 83.0 5.8 87.2 5.4 84.4 3.1 25 85.4 9.2 92.1 7.9 102.6 9.2 93.4 6.9 90.3 2.0 10 103.9 6.3 103.2 10.1 102.2 8.1 93.6 7.0 106.0 2.6 One 103.1 12.3 101.6 8.2 97.8 6.2 103.2 14.9 117.7 7.4 0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 DMSO 1% 94.7 6.2 94.7 6.2 94.7 6.2 94.7 6.2 94.7 6.2 SDS 5% 44.1 8.0 44.1 8.0 44.1 8.0 44.1 8.0 44.1 8.0 THP-1 100 68.1 0.9 70.8 4.1 54.3 1.7 74.9 3.7 98.8 9.5 50 79.5 3.2 78.2 8.4 75.5 4.1 78.8 4.1 99.1 10.9 25 74.5 3.1 80.0 4.9 75.6 5.0 79.1 5.8 92.4 8.6 10 75.9 3.7 78.9 5.1 75.0 3.1 81.3 3.2 93.2 7.5 One 75.7 1.8 80.4 3.5 82.7 5.9 87.0 7.8 93.9 5.6 0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 DMSO 1% 103.3 6.2 103.3 6.2 103.3 6.2 103.3 6.2 103.3 6.2 SDS 5% 36.2 4.6 36.2 4.6 36.2 4.6 36.2 4.6 36.2 4.6 CCD1079SK 100 91.9 4.5 93.3 8.8 60.6 8.4 68.1 6.7 96.6 4.1 50 102.4 8.2 103.0 12.1 85.6 8.6 76.8 6.0 100.1 4.5 25 99.3 1.5 101.8 11.5 85.8 13.8 81.2 3.4 98.8 6.2 10 100.7 5.1 99.5 13.5 93.8 8.1 84.0 1.5 96.9 5.1 One 102.7 4.4 98.3 9.8 97.3 10.9 89.2 4.7 98.5 1.9 0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 DMSO 1% 87.2 9.7 87.2 9.7 87.2 9.7 87.2 9.7 87.2 9.7 SDS 5% 11.3 0.6 11.3 0.6 11.3 0.6 11.3 0.6 11.3 0.6

IC for NC77 fraction ₅₀  Calculation NC77 F1 F2 F3 F4 F5 SKOV3 - - - 80.2 - MCF7 - - 67.9 73.9 - U373 - - - - - THP-1 - - - - - CCD1079SK - - - - -

LD ₅₀ > 1-μg / ml

Example 4 Extraction # 1 (NC77) from fraction 4

Fraction 4 was selected for further subfractionation based on the biological quantification results. (Table 5) Thus, in the second step, NC77-F4 was dissolved in dichloromethane / methanol, mixed with celite, and dried by rotary evaporation. Samples of de-Reda GOLD ISCO high-performance silica gel was loaded on a column and 24g, eluting with dichloromethane / methanol on CombiFIash ^® Rf, deionized Reda the ISCO. Subfractionation was carried out as follows for the rate of increase / decrease of solvent A (CH ₂ CI ₂ ) and solvent B (1: 1 methanol: water). 0% B for 2CV (column volume), 40% B for 17CV, 100% B for 4CV, total elution at 23CV

The fractions were monitored by TLC and the fractions were combined and dried using Rotavap and Genevac in the yield 5 sub-fraction as shown in Table 7.

The lower fractionation yield of NC77-F4 Sample Sample
Weight (g) Fraction 1 (g) Fraction 2 (g) Fraction 3 (g) Fraction 4 (g) Fraction 5 (g) NC77 - fraction 4 0.21 0.011 0.044 0.006 0.036 0.064

These subfractions were again subjected to the biological quantitative evaluation presented above. The% viability (method + standard deviation three times repeated verification) together with the LD50 / lC50 values in Table 9 are shown in Table 8. [ The most active results are displayed in gray and statistical analysis results are shown. The maximum DMSO concentration was 1% at 100 μg / ml with 5% SDS used as a known toxic inducer.

The results clearly showed that the anticancer activity was distributed from fraction 4 of NC77 to the lower fractions F3 and F4.

In vitro screening results - NC77 - subfraction from fraction 4 NC77
Cell line (ug / ml) F1 F2 F3 F4 F5 Average Standard Average Standard Average Standard Average Standard Average Standard PC3 100 104.4 3.4 92.4 4.2 89.7 6.8 98.9 6.0 87.9 2.6 50 116.2 21.2 93.3 7.3 93.3 8.3 93.2 6.1 89.3 3.9 25 120.9 13.8 97.6 8.6 97.9 9.0 98.4 1.9 95.0 4.5 10 115.4 9.5 99.6 8.5 99.3 6.1 101.4 3.5 96.1 6.0 One 120.9 14.2 96.7 10.9 101.7 9.9 101.6 8.0 105.5 3.4 0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 DMSO 1% 7.7 0.8 7.7 0.8 7.7 0.8 7.7 0.8 7.7 0.8 SDS 5% 94.4 6.1 94.4 6.1 94.4 6.1 94.4 6.1 94.4 6.1 SKOV3 100 91.0 8.2 91.2 4.4 86.6 1.9 92.0 8.0 84.6 2.6 50 97.7 0.8 102.8 2.2 95.3 2.1 101.7 3.9 86.7 2.6 25 100.0 1.3 103.3 1.7 100.0 4.5 103.2 2.0 86.1 4.5 10 99.0 3.8 102.2 0.7 99.8 0.9 101.2 4.0 91.0 1.6 98.1 1.3 103.6 2.6 100.5 6.5 99.7 1.0 97.0 3.6 0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 DMSO 1% 14.3 0.7 14.3 0.7 14.3 0.7 14.3 0.7 14.3 0.7 SDS 5% 90.7 33.4 90.7 33.4 90.7 33.4 90.7 33.4 90.7 33.4 A549 100 100.1 11.7 109.9 12.7 88.7 12.2 98.2 11.1 88.2 5.4 50 107.9 3.7 103.3 10.5 99.7 10.7 110.6 2.3 83.0 4.6 25 110.8 5.4 105.9 5.1 104.6 9.5 117.5 3.7 81.6 7.1 10 112.9 2.1 109.5 5.0 106.3 9.6 117.1 12.1 94.5 11.9 One 106.1 4.0 112.8 12.5 105.0 6.0 117.4 5.6 94.7 8.7 0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 DMSO 1% 6.4 0.6 6.4 0.6 6.4 0.6 6.4 0.6 6.4 0.6 SDS 5% 66.2 13.3 66.2 13.3 66.2 13.3 66.2 13.3 66.2 13.3 THP-1 100 11.0 0.5 12.5 1.2 15.0 1.6 27.2 4.0 19.1 2.0 50 37.5 1.9 39.3 1.1 26.9 3.6 57.1 4.7 59.8 8.6 25 61.9 1.4 71.8 5.9 66.5 8.3 67.5 2.2 85.5 5.5 10 99.1 5.1 78.0 8.9 72.9 9.2 76.3 4.5 87.9 6.1 One 102.2 10.6 96.8 8.6 104.3 12.7 78.2 7.2 96.5 5.0 0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 DMSO 1% 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 SDS 5% 39.9 2.9 39.9 2.9 39.9 2.9 39.9 2.9 39.9 2.9 U373 100 6.0 1.4 3.3 0.8 1.4 0.5 28.1 3.5 5.3 0.7 50 79.6 8.4 109.0 14.2 43.4 7.0 67.4 4.5 61.5 9.4 25 92.5 30.1 113.0 2.9 88.2 5.5 72.1 2.0 66.0 8.5 10 108.4 18.5 102.6 9.6 103.5 13.8 76.1 4.1 88.7 8.2 One 106.1 14.8 117.6 5.4 110.5 7.3 95.9 3.0 81.0 9.5 0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 DMSO 1% 3.0 0.4 3.0 0.4 3.0 0.4 3.0 0.4 3.0 0.4 SDS 5% 78.5 11.6 78.5 11.6 78.5 11.6 78.5 11.6 78.5 11.6 CCD1079SK 100 22.5 3.1 8.2 0.9 9.4 1.9 32.9 1.8 66.6 13.3 50 54.2 0.5 89.4 6.7 9.3 3.5 60.3 4.6 82.6 6.1 25 89.8 1.8 95.5 2.7 58.1 5.2 81.1 8.6 76.3 7.5 10 94.0 4.7 98.1 11.2 85.2 8.9 86.2 4.7 98.7 11.6 One 101.5 4.8 95.6 0.9 101.7 8.7 98.8 6.9 96.3 4.1 0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 DMSO 1% 8.6 0.9 8.6 0.9 8.6 0.9 8.6 0.9 8.6 0.9 SDS 5% 87.9 7.2 87.9 7.2 87.9 7.2 87.9 7.2 87.9 7.2 MCF7 100 23.1 2.9 24.1 0.5 26.7 2.0 25.1 1.7 57.5 7.3 50 42.3 3.7 69.3 3.2 56.4 8.1 45.2 4.6 103.6 12.1 25 83.7 9.0 92.5 2.3 84.3 8.4 81.8 5.1 108.6 4.0 10 93.6 7.2 91.4 1.2 101.0 11.8 84.3 0.5 104.3 6.8 One 96.7 2.6 98.6 6.6 98.7 9.0 87.3 3.3 107.3 4.7 0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 DMSO 1% 16.9 3.3 16.9 3.3 16.9 3.3 16.9 3.3 16.9 3.3 SDS 5% 93.7 8.7 93.7 8.7 93.7 8.7 93.7 8.7 93.7 8.7

LD of NC77-F4 (占 퐂 / ml) ₅₀  Calculation subfraction Cell line (ug / ml) F1 F2 F3 F4 F5 PC3 - - - - - SKOV3 - - - - - A549 - - - - - THP-1 31.1 48.8 34.0 58.8 62.3 U373 68.6 - 49.1 66.9 50.4 CCD1079SK 64.3 - 22.2 71.1 - MCF7 59.4 70.4 58.1 50.6 -

Example 5 Principal component characteristics of NC77 and NC77-F4

5.1. Sample preparation and analysis

FAME production and GC-MS analysis

Extract # 1 (NC77) and its fraction-4 (NC77-Fr. 4) were analyzed by GC-MS. The sample was capped and 1 mL of sodium methoxide solution and 1 mL of hexane were added. The reaction vial is placed in a heating block (80 ° C) and shaken by hand for 15 minutes at 5 minute intervals. After cooling to room temperature, 1 mL of saturated NaCl solution, which had been shaken by hand several times, was added. The reaction vial was then centrifuged at 2,000 rpm for 20 minutes. The upper solution was transferred to a GC vial. For GC-MS analysis, Agilent 6890 with mass spectrometry detector was used. Columns are Agilent DB-23 (59m Х 0.25mm, 0.15μm) and injection volume is 1μL. The oven program was 1 minute at 50 占 폚, 170 占 폚 at 25 占 폚 / min, 215 占 폚 (holding for 12 minutes) at 2.75 占 폚 / min, and 230 占 폚 at 40 占 min. The total run time was 37.65 minutes, the FID temperature was 280 DEG C, the hydrogen flow rate was 40 mL / min, the air flow rate was 400 mL / min, and the feed flow rate N2 was 20 mL / min. The split ratio was 2: 1. The carrier gas was helium and maintained a constant pressure (30 psi). The MSD ionization mode was EI. The interface temperature was 250 占 폚, the MS source was 230 占 폚, MS quad 150 占 폚, and the mass range was 50-600 m / z.

HPLC-DAD analysis for comparing different batches

Separation was carried out on an Agilent Zorbax SB-C18 (2.1 x 30 mm 3.5 pm) column using Agilent HPLC 1100. Solvent A was 10 mM ammonium formate (pH 3.2) and solvent B was 90% acetonitrile with 10% 100 mM ammonium formate (pH 3.2). The rate of increase was 100% B at 30% B for 12 minutes and washed with 0.1% formic acid for 2 minutes in acetonitrile. The column temperature was 55 占 폚. The flow rate was 0.5 mL / min.

HPLC-DAD / MS analysis of carotenoids

Analysis was performed at 32 째 C using a YMC carotinoid column on an Agilent 1200 system at 0.5 袖 m (250 x 2 mm). Solvent 50 mM DMAc / MeOH, solvent B MTBE, 5-65% B increase / decrease within 40 minutes. The flow rate was 0.2 mL / min and the DAD detector was examined at 450 nm. Peak confirmation in chromatograms was based on RT comparisons to known standards such as fucosanthin, astaxanthin, lutein, zeaxanthin, canthaxanthin, alpha and beta carotene. All standards were purchased from Chromadex.

UPLC-DAD / ELSD / HRMS and MS / MS

The following instrument was used for LC-UV-ELSD-HRMS data collection. Accela 1250 pump (Thermo Fisher Scientific), Exactive benchtop Orbitrap mass spectrometer (Thermo Fisher), Utimate 3000 DAD (Thermo Scientific Dionex) and ELSD 3300 (Alltech) equipped with a heated electrospray ionization probe. Separation was carried out using a mobile phase consisting of (A) 0.1% formic acid and (B) 0.1% formic acid in acetonitrile with a linear gradient from 5% B to 100% C18 column (50X2.1 mm, Thermo), and the flow rate was 400 L / min.

HRMS was obtained with positive or negative polarity at 25,000 resolution and HCD scans were used with a collision energy of 50 eV for all ion fragments each with a resolution of 10,000. The following optimal ion source conditions were used. The external flow rate is 15 and the auxiliary gas flow rate is 3. Spray voltage 3kV (-2.5kV for negative electrode). The capillary and heater temperatures are 350 ° C and 250 ° C, respectively.

NMR

Samples were reconstituted in 100 μl of CDCl ₃ and 60 μl of each sample was transferred to a 1.7 mm NMR tube. All spectra were measured on a Bruker Avance III 700 MHz spectrometer equipped with a 1.7 mm cryogenic cooling probe operating at 16K.

5.2. Analysis of NC77 and NC77-Fr 4

Previously conducted preliminary information collection work indicated that the lipid is the major component of NC77-Fr.4. As such, GC-MS analysis was performed to determine the fatty acid composition of the extract and its bioactive fractions.

Figure 5 is a GC chromatogram of FAME prepared from extract # 1 (NC77) and # 1-F4 (NC77-Fr. 4). Time spent and uncertainty based on NIST database matches are shown in Table 10. The major fatty acid was palmitic acid.

In addition, NC77 and NC77-Fr. 4 profile NMR and HPLC of two different batches were compared.

6 and 7, extract # 1 and fraction 4 (NC77 and NC77-Fr. 4) prepared in November 2015 were similar to those observed in the initial batch (June 2015) NMR profile. Lipid (fatty acid) was the main component.

The HPLC comparison is shown in FIG. The two seem to have a similar profile of the main component.

Example 6 Fractionation and Purification of the Major Component from NC77-F4-F13

In a second series of experiments, NC77 was fractionated as described above and fraction 4 was subfractionated but slightly different protocol was used to obtain better compound separation. In summary, 3.14 g of Extract # 1 (NC77) was dissolved in methanol, mixed on celite and dried using a rotary evaporator. The sample was then loaded into a Thermo Scientific SPE column (HYPER SEP C18 20G) maintained in a pre-conditioned and balanced state. SPE column was dissolved with 5% methanol (fraction 1), 25% methanol (fraction 2), 50% methanol (fraction 3) and 100% methanol (fraction 4) to obtain four fractions.

Based on the previous biochemical results, fraction-4 (0.34 g) was further fractionated. Fraction 4 was dissolved in dichloromethane / methanol, mixed with celite and dried. Samples of de-Reda GOLD ISCO high-performance silica gel was loaded on a column and 24g, eluting with dichloromethane / methanol on CombiFIash ^® Rf, deionized Reda the ISCO. Solvent extraction rates (A and B) are as follows. 0% B for 2CV (column volume), 40% B for 15CV and 40% B for 2CV, 100% B for 2CV and 100% B for 2CV. The total elution amount was 23 CV. A is dichloromethane and B is methanol / dichloromethane (1: 1). The fractions were monitored by TLC and some were combined and dried using Rotavap and Genevac.

Based on TLC analysis, the lower fraction 13 of fraction 4 appeared to contain the main component and was selected for further purification. At this stage, CombiFlash ^® Rf used 12 g silica gel column. The rate of change of the solvents (A and B) is as follows. 0% B for 2CV, 100% B for 25CV and 100% B for 2CV. Total elution was 29 CV. A is dichloromethane and B is 5% methanol in dichloromethane. Again, the fractions were selectively coupled according to TLC.

The three sub-fractions were further purified using semi-purified HPLC (Agilent). The column used was ZORBAX SB-C18 (9.4 x 50 mm, 5 m) and the mobile phase was water / acetonitrile. Since the rate of change in solvent varies from sample to sample, the separation can be optimized. The column temperature was 55 DEG C and the flow rate was 5 mL / min.

As shown in FIG. 9, the above fractionation and purification yielded 19 samples for complete structural analysis. Among them, Sample 31-34-5 (3.9 mg) was a pure compound.

Example 7 Structure of pure compound 31-34-5

7.1. Compound 31-34-5 (formula (I))

UPLC-DAD / ELSD / HRMS data (Figure 10) indicated compound 31-34-5 as pure compound. HRMS (Fig. 11) m / z 335.21950 reached a peak of (positive mode, C ₁₈ H ₃₁ O ₄ observed for Na ^+, calculated 335.21983), m / z311.22283 (negative mode, C ₁₈ H ₃₁ O ₄ ^- , observed as 311.22223) confirmed the molecular formula of C ₁₈ H ₃₂ O ₄ .

^{The 1} H-NMR, COZY, TOCSY and HSQC spectra (15 in FIG. 12) show two olefin methines (delta 5.79 ppm), three oxidases (delta 5.34, 4.09, 3.67 ppm), 11 methylene -1.0 ppm) and a 1-terminal methyl group (0.87 ppm).

HMBC (Figure 16) revealed a major C-H correlation suggesting 12,13-dihydroxy-10-octadecen-9-olide as the structure for compound 31-34-5. (Arrows for major HMBC correlations)

(I).

(I).

The double bonds at C-10, 11 were determined to be trans forms based on the coupling value J of the olefin protons. HRMS (Fig. 17) Sculpture ions also confirmed its structure.

Based on the database structure search, compound 31-34-5 is a novel compound. It is an unsaturated esterification and a fatty acid derivative which is likely to be formed via octadecanoic acid or stearic acid (18: 0). The full spectral data arrangement is listed in Table 11.

NMR data of Compound 31-34-5 location dC, ppm Kinds dH, ppm (J in Hz) One 175.3 C = O 2 36.7 CH ₂ 2.51 ddd (15.9, 6.4, 3.3)
2.18 (13.6, 2.8) 3 21.7 CH ₂ 2.04m
1.54m 4 28.2 CH ₂ 1.45m 5 25.3 CH ₂ 1.54m
1.36m 6 24.9 CH ₂ 1.45m
1.02m 7 24.7 CH ₂ 1.70m
1.23m 8 31.4 CH ₂ 1.97m
1.55m 9 76.7 CH 5.34m 10 132.6 CH 5.78dd (15.9; 4.6) 11 130.4 CH 5.76dd (15.9; 5.0) 12 76.2 CH 4.08br.s 13 75.3 CH 3.64br.s 14 33.1 CH ₂ 1.37m 15 26.8 CH ₂ 1.47m
1.29m 16 32.9 CH ₂ 1.29m 17 23.6 CH ₂ 1.29m 18 14.7 CH ₃ 0.87t (7.1)

Example 8 Compound of In Vitro Anticancer Activity Formula (I)

Compound (I) of the in vitro anticancer activity is shown as average cell viability in Table 12, while Table 13 shows the same data, but shows the survival rate ± standard error of doubling.

Mediator cont-DMSO One% 96 86 98 93 92 91 88 Positive cont-SDS 250ug / ml 8 11 4 9 14 5 16 Sample
# Sample ID Ug / mL PC3 A549 U373 SKOV MDA-
MB CCD THP-1

3

31-34-5 100 19 61 3 85 20 17 44 50 97 92 68 94 66 90 68 10 93 93 85 97 96 84 84 One 100 95 90 97 101 91 96

The invention has been described in terms of specific embodiments found or suggested by the inventors to include preferred modes for their implementation. In light of the present fact, it will be appreciated by persons skilled in the art that many modifications and variations can be made in the specific example that is illustrated without departing from the intended scope of the invention. All such modifications are intended to be included within the scope of the appended claims.

All publications and patent applications cited in this specification are incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

bis-AAF-R110: bis-alanyl-alanyl-phenylalanyl-rhodamine 110
CIMA: color indicating target activity
GF-AFC: Gly-Phe-7-amino-4-trifluoromethylcoumarin
HlLlC: Hydrophilic Interaction Liquid Chromatography
C-18 SPE: Extraction of solid state in C-18 column

Claims (28)

The compound of formula (I)

(I). The method according to claim 1,
Racemic mixture or a compound as an enantiomer. 3. The method according to claim 1 or 2,
Use of compounds to inhibit the growth of cancer cells in vitro. 3. The method according to claim 1 or 2,
Use of the compound to inhibit cancer cell growth in a mammal. 3. The method according to claim 1 or 2,
A compound for use in the treatment or prevention of cancer in a mammal. 3. The method according to claim 1 or 2,
Use of a compound for the manufacture of a composition for treating or preventing cancer in a mammal. 3. The method according to claim 1 or 2,
Compounds integrated into oral preparations. 8. The method of claim 7,
Wherein said oral preparation is a neuraminidase or a nutritional formulation. A seaweed extract concentrated in a compound of formula (I).

(I). 10. The method of claim 9,
Use of the extract to inhibit the growth of cancer cells in vitro. 10. The method of claim 9,
Use of said extract to inhibit cancer cell growth in a mammal. 10. The method of claim 9,
Said extract for use in the treatment or prevention of cancer in a mammal. 10. The method of claim 9,
The use of said extract for the manufacture of a composition for treating or preventing cancer in a mammal. 10. The method of claim 9,
Said extract being incorporated into an oral preparation. 15. The method of claim 14,
Wherein the oral preparation is a Nutraceutical or a nutritional formulation. 9. A composition according to claim 1 or 2 or an extract according to claim 9 mixed with a physiologically acceptable additive. 17. The method of claim 16,
Wherein the additive is an orally acceptable additive. 18. The method according to claim 16 or 17,
A composition for use in the treatment or prevention of cancer in a mammal. 18. The method according to claim 16 or 17,
Use of the composition for use in the treatment or prevention of cancer in a mammal. 18. The method of claim 17,
An oral composition in combination with one or more other therapeutic agents. 21. The method of claim 20,
Wherein the other therapeutic agent is an anti-cancer agent. A powder, syrup, gelatin capsule, tablet, capsule or other device for oral administration comprising an anti-cancer effective amount of a compound according to claims 1 or 2 or an extract according to claim 9. In cancer treatment methods,
A method of treating cancer comprising administering to said mammal a compound according to any one of claims 1 or 2, an extract according to claim 9 or a composition according to claim 16 for inhibiting the growth of cancer cells. 24. The method of claim 23,
Wherein the mammal is a pet animal or a human. a) Chaetomorpha Cannabina CC to obtain biomass (ethanol mixture); 80% aqueous ethanol mixing of biomass of seaweed;
b) eluting the mixture in an SPE column with varying concentrations of methanol, collecting 100% methanol fractions;
c) fractionating the methanol fraction from step b) on a Combiflash column with various concentrations of MeOH: CH ₂ Cl ₂ , and recovering the fraction containing the major component. 26. The method of claim 25,
d) subclassifying said fraction with water / acetonitrile to obtain a sub-fraction on a semi-preparative HPLC column. 27. The method of claim 26,
e) drying the lower fraction by removing the solvent to obtain a concentrated dry extract in the compound of formula (I)

(I). 27. The method of claim 26,
d ') further comprising purifying said compound of formula (I) from said lower fraction to obtain a purified compound of formula (I).

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